The demand for efficient and large scale production of therapeutic proteins is steadily increasing as more recombinant proteins are approved for use in humans. Using E. coli expression system for production of recombinant proteins, however, frequently results in formation of water-insoluble protein inclusion bodies, instead of functional, soluble proteins. Additionally, resolubilized proteins from inclusion bodies do not elicit as strong an immune responses in rats and mice as soluble, native protein does.
The Hsp70-peptide antigen complex plays an important role in the antigen presentation process (Mycko et al., 2004; Bendz et al., 2007). The molecular chaperone/co-chaperone pair (Hsp70/Hsp40) is highly conserved throughout evolution. Eukaryotic Hsp70 genes are descents of the bacterial DnaK gene. DnaK and HSP70 have over 50% homology in their amino acid sequences. The structure of DnaK/Hsp70 is composed of a N-terminal ATPase domain, a substrate binding domain, and a C-terminal lid domain (Genevaux et al., 2007; Shaner & Morano, 2007). Hsp40s represent a large protein family that functions to specify the cellular action of Hsp70 chaperone proteins. There are 44 members of Hsp40 genes found in human genome. Twelve of them are descents of E. coli DnaJ. Although their amino acid sequences outside the J-domain are divergent, the 75 amino acid J-domain of the Hsp40 proteins is highly conserved. The J-domain of Hsp40 interacts directly with the Hsp70 ATPase domain to enhance the ATP hydrolysis rate which in turn increases the substrate binding affinity. Hsp40 proteins also bind client substrate and deliver it to the Hsp70 (Fan et al., 2003).
Protein transduction domain (PTD) peptides are able to ferry large molecules into cells independent of classical endocytosis. They are used to enhance cellular uptake of drugs, proteins, polynucleotides and liposomes (Tilstra et al., 2007).
Neither the PTD nor the Hsp40 J-domain has been employed in the development of an expression system for improving solubility and immunogenicity of a recombinant protein. A previously unaddressed need exists in the art to address the aforementioned deficiencies and inadequacies, especially in connection with the development of an expression system for a recombinant protein with enhanced solubility and immunogenicity.